1. Field of the Invention
The present invention relates to methods and apparatus for mass spectrometry.
2. Discussion of the Prior Art
Tandem mass spectrometry (MS/MS) is the name given to the method of mass spectrometry wherein parent ions generated from a sample are selected by a first mass filter/analyser and are then passed to a collision cell wherein they are fragmented by collisions with neutral gas molecules to yield daughter (or xe2x80x9cproductxe2x80x9d) ions. The daughter ions are then mass analysed by a second mass filter/analyser, and the resulting daughter ion spectra can be used to determine the structure and hence identify the parent (or xe2x80x9cprecursorxe2x80x9d) ion. Tandem mass spectrometry is particularly useful for the analysis of complex mixtures such as biomolecules since it avoids the need for chemical clean-up prior to mass spectral analysis.
A particular form of tandem mass spectrometry referred to as parent ion scanning is known, wherein in a first step the second mass filter/analyser is arranged to act as a mass filter so that it will only transmit and detect daughter ions having a specific mass-to-charge ratio. The specific mass-to-charge ratio is set so as to correspond with the mass-to-charge ratio of daughter ions which are known to be characteristic products which result from the fragmentation of a particular parent ion or type of parent ion. The first mass filter/analyser upstream of the collision cell is then scanned whilst the second mass filter/analyser remains fixed to monitor for the presence of daughter ions having the specific mass-to-charge ratio. The parent ion mass-to-charge ratios which yield the characteristic daughter ions can then be determined. As a second step, a complete daughter ion spectrum for each of the parent ion mass-to-charge ratios which produce characteristic daughter ions may then be obtained by operating the first mass filter/analyser so that it selects parent ions having a particular mass-to-charge ratio, and scanning the second mass filter/analyser to record the resulting full daughter ion spectrum. This can then be repeated for the other parent ions of interest. Parent ion scanning is useful when it is not possible to identify parent ions in a direct mass spectrum due to the presence of chemical noise, which is frequently encountered, for example, in the electrospray mass spectra of biomolecules.
Triple quadrupole mass spectrometers having a first quadrupole mass filter/analyser, a quadrupole collision cell into which a collision gas is introduced, and a second quadrupole mass filter/analyser are well known. Another type of mass spectrometer (a hybrid quadrupole-time of flight mass spectrometer) is known wherein the second quadrupole mass filter/analyser is replaced by an orthogonal time of flight mass analyser.
As will be shown below, both types of mass spectrometers when used to perform conventional methods of parent ion scanning and subsequently obtaining a daughter ion spectrum of a candidate parent ion suffer from low duty cycles which render them unsuitable for use in applications which require a higher duty cycle such as on-line chromatography applications.
Quadrupoles have a duty cycle of approximately 100% when being used as a mass filter, but their duty cycle drops to around 0.1% when then are used in a scanning mode as a mass analyser, for example, to mass analyse a mass range of 500 mass units with peaks one mass unit wide at their base.
Orthogonal acceleration time of flight analysers typically have a duty cycle within the range 1-20% depending upon the relative mass to charge (xe2x80x9cm/zxe2x80x9d) values of the different ions in the spectrum. However, the duty cycle remains the same irrespective of whether the time of flight analyser is being used as a mass filter to transmit ions having a particular mass to charge ratio, or whether the time of flight analyser is being used to record a full mass spectrum. This is due to the nature of operation of time of flight analysers. When used to acquire and record a daughter ion spectrum the duty cycle of a time of flight analyser is typically around 5%.
To a first approximation the conventional duty cycle when seeking to discover candidate parent ions using a triple quadrupole mass spectrometer is approximately 0.1% (the first quadrupole mass filter/analyser is scanned with a duty cycle of 0.1% and the second quadrupole mass filter/analyser acts as a mass filter with a duty cycle of 100%). The duty cycle when then obtaining a daughter ion spectrum for a particular candidate parent ion is also approximately 0.1% (the first quadrupole mass filter/analyser acts as a mass filter with a duty cycle of 100%, and the second quadrupole mass filter/analyser is scanned with a duty cycle of approximately 0.1%). The resultant duty cycle therefore of discovering a number of candidate parent ions and producing a daughter spectrum of one of the candidate parent ions is approximately 0.1%/2 (due to a two stage process with each stage having a duty cycle of 0.1%)=0.05%.
The duty cycle of a quadrupole-time of flight mass spectrometer for discovering candidate parent ions is approximately 0.005% (the quadrupole is scanned with a duty cycle of approximately 0.1% and the time of flight analyser acts a mass filter with a duty cycle of approximately 5%). Once candidate parent ions have been discovered, a daughter ion spectrum of a candidate parent ion can be obtained with an duty cycle of 5% (the quadrupole acts as a mass filter with a duty cycle of approximately 100% and the time of flight analyser is scanned with a duty cycle of 5%). The resultant duty cycle therefore of discovering a number of candidate parent ions and producing a daughter spectrum of one of the candidate parent ions is approximately 0.005% (since 0.005% less than  less than 5%).
As can be seen, a triple quadrupole has approximately an order higher duty cycle than a quadrupole-time of flight mass spectrometer for performing conventional methods of parent ion scanning and obtaining confirmatory daughter ion spectra of discovered candidate parent ions. However, such duty cycles are not high enough to be used practically and efficiently for analysing real time data which is required when the source of ions is the eluent from a chromatography device.
Electrospray and laser desorption techniques have made it possible to generate molecular ions having very high molecular weights, and time of flight mass analysers are advantageous for the analysis of such large mass biomolecules by virtue of their high efficiency at recording a full mass spectrum. They also have a high resolution and mass accuracy.
Other forms of mass analysers such as quadrupole ion traps are similar in some ways to time of flight analysers, in that like time of flight analysers, they can not provide a continuous output and hence have a low efficiency if used as a mass filter to continuously transmit ions which is an important feature of the conventional methods of parent ion scanning. Both time of flight mass analysers and quadrupole ion traps may be termed xe2x80x9cdiscontinuous output mass analysersxe2x80x9d.
It is desired to provide improved methods and apparatus for mass spectrometry. In particular, it is desired to identify parent ions in chromatography applications.
Parent ions that belong to a particular class of parent ions, and which are recognisable by a characteristic daughter ion or characteristic xe2x80x9cneutral lossxe2x80x9d, are traditionally discovered by the methods of xe2x80x9cparent ionxe2x80x9d scanning or xe2x80x9cconstant neutral lossxe2x80x9d scanning. Previous methods for recording xe2x80x9cparent ionxe2x80x9d scans or xe2x80x9cconstant neutral lossxe2x80x9d scans involve scanning one or both quadrupoles in a triple quadrupole mass spectrometer, or scanning the quadrupole in a tandem quadrupole orthogonal TOF mass spectrometer, or scanning at least one element in other types of tandem mass spectrometers. As a consequence, these methods suffer from the low duty cycle associated with scanning instruments. As a further consequence, information may be discarded and lost whilst the mass spectrometer is occupied recording a xe2x80x9cparent ionxe2x80x9d scan or a xe2x80x9cconstant neutral lossxe2x80x9d scan. As a further consequence these methods are not appropriate for use where the mass spectrometer is required to analyse substances eluting directly from gas or liquid chromatography equipment.
According to the preferred embodiment, a tandem quadrupole orthogonal TOF mass spectrometer is used in a way in which candidate parent ions are discovered using a method in which sequential low and high collision energy mass spectra are recorded. The switching back and forth is not interrupted. Instead a complete set of data is acquired, and this is then processed afterwards. Fragment ions are associated with parent ions by closeness of fit of their respective elution times. In this way candidate parent ions may be confirmed or otherwise without interrupting the acquisition of data, and information need not be lost.
Once an experimental run has been completed, the high and low fragmentation mass spectra are then post-processed. Parent ions are recognised by comparing a high fragmentation mass spectrum with a low fragmentation mass spectrum obtained at substantially the same time, and noting ions having a greater intensity in the low fragmentation mass spectrum relative to the high fragmentation mass spectrum. Similarly, daughter ions may be recognised by noting ions having a greater intensity in the high fragmentation mass spectrum relative to the low fragmentation mass spectrum.
Once a number of parent ions have been recognised, a sub-group of possible candidate parent ions may be selected from all of the parent ions. According to one embodiment, possible candidate parent ions may be selected on the basis of their relationship to a predetermined daughter ion. The predetermined daughter ion may comprise, for example, ions selected from the group comprising: (i) immonium ions from peptides; (ii) functional groups including phosphate group PO3 ions from phosphorylated peptides; and (iii) mass tags which are intended to cleave from a specific molecule or class of molecule and to be subsequently identified thus reporting the presence of the specific molecule or class of molecule. A parent ion may be short listed as a possible candidate parent ion by generating a mass chromatogram for the predetermined daughter ion using high fragmentation mass spectra. The centre of each peak in the mass chromatogram is then determined together with the corresponding predetermined daughter ion elution time(s). Then for each peak in the predetermined daughter ion mass chromatogram both the low fragmentation mass spectrum obtained immediately before the predetermined daughter ion elution time and the low fragmentation mass spectrum obtained immediately after the predetermined daughter ion elution time are interrogated for the presence of previously recognised parent ions. A mass chromatogram for any previously recognised parent ion found to be present in both the low fragmentation mass spectrum obtained immediately before the predetermined daughter ion elution time and the low fragmentation mass spectrum obtained immediately after the predetermined daughter ion elution time is then generated and the centre of each peak in each mass chromatogram is determined together with the corresponding possible candidate parent ion elution time(s). The possible candidate parent ions may then be ranked according to the closeness of fit of their elution time with the predetermined daughter ion elution time, and a list of final candidate parent ions may be formed by rejecting possible candidate parent ions if their elution time precedes or exceeds the predetermined daughter ion elution time by more than a predetermined amount.
According to an alternative embodiment, a parent ion may be shortlisted as a possible candidate parent ion on the basis of it giving rise to a predetermined mass loss. For each low fragmentation mass spectrum, a list of target daughter ion mass to charge values that would result from the loss of a predetermined ion or neutral particle from each previously recognised parent ion present in the low fragmentation mass spectrum is generated. Then both the high fragmentation mass spectrum obtained immediately before the low fragmentation mass spectrum and the high fragmentation mass spectrum obtained immediately after the low fragmentation mass spectrum are interrogated for the presence of daughter ions having a mass to charge value corresponding with a target daughter ion mass to charge value. A list of possible candidate parent ions (optionally including their corresponding daughter ions) is then formed by including in the list a parent ion if a daughter ion having a mass to charge value corresponding with a target daughter ion mass to charge value is found to be present in both the high fragmentation mass spectrum immediately before the low fragmentation mass spectrum and the high fragmentation mass spectrum immediately after the low fragmentation mass spectrum. A mass loss chromatogram may then be generated based upon possible candidate parent ions and their corresponding daughter ions. The centre of each peak in the mass loss chromatogram is determined together with the corresponding mass loss elution time(s). Then for each possible candidate parent ion a mass chromatogram is generated using the low fragmentation mass spectra. A corresponding daughter ion mass chromatogram is also generated for the corresponding daughter ion. The centre of each peak in the possible candidate parent ion mass chromatogram and the corresponding daughter ion mass chromatogram are then determined together with the corresponding possible candidate parent ion elution time(s) and corresponding daughter ion elution time(s). A list of final candidate parent ions may then be formed by rejecting possible candidate parent ions if the elution time of a possible candidate parent ion precedes or exceeds the corresponding daughter ion elution time by more than a predetermined amount.
Once a list of final candidate parent ions has been formed (which preferably comprises only some of the originally recognised parent ions and possible candidate parent ions) then each final candidate parent ion can then be identified.
Identification of parent ions may be achieved by making use of a combination of information. This may include the accurately determined mass of the parent ion. It may also include the masses of the fragment ions. In some instances the accurately determined masses of the daughter ions may be preferred. It is known that a protein may be identified from the masses, preferably the exact masses, of the peptide products from proteins that have been enzymatically digested. These may be compared to those expected from a library of known proteins. It is also known that when the results of this comparison suggest more than one possible protein then the ambiguity can be resolved by analysis of the fragments of one or more of the peptides. The preferred embodiment allows a mixture of proteins, which have been enzymatically digested, to be identified in a single analysis. The masses, or exact masses, of all the peptides and their associated fragment ions may be searched against a library of known proteins. Alternatively, the peptide masses, or exact masses, may be searched against the library of known proteins, and where more than one protein is suggested the correct protein may be confirmed by searching for fragment ions which match those to be expected from the relevant peptides from each candidate protein.
The step of identifying each final candidate parent ion preferably comprises: recalling the elution time of the final candidate parent ion, generating a list of possible candidate daughter ions which comprises previously recognised daughter ions which are present in both the low fragmentation mass spectrum obtained immediately before the elution time of the final candidate parent ion and the low fragmentation mass spectrum obtained immediately after the elution time of the final candidate parent ion, generating a mass chromatogram of each possible candidate daughter ion, determining the centre of each peak in each possible candidate daughter ion mass chromatogram, and determining the corresponding possible candidate daughter ion elution time(s). The possible candidate daughter ions may then be ranked according to the closeness of fit of their elution time with the elution time of the final candidate parent ion. A list of final candidate daughter ions may then be formed by rejecting possible candidate daughter ions if the elution time of the possible candidate daughter ion precedes or exceeds the elution time of the final candidate parent ion by more than a predetermined amount.
The list of final candidate daughter ions may be yet further refined or reduced by generating a list of neighbouring parent ions which are present in the low fragmentation mass spectrum obtained nearest in time to the elution time of the final candidate parent ion. A mass chromatogram of each parent ion contained in the list is then generated and the centre of each mass chromatogram is determined along with the corresponding neighbouring parent ion elution time(s). Any final candidate daughter ion having an elution time which corresponds more closely with a neighbouring parent ion elution time than with the elution time of the final candidate parent ion may then be rejected from the list of final candidate daughter ions.
Final candidate daughter ions may be assigned to a final candidate parent ion according to the closeness of fit of their elution times, and all final candidate daughter ions which have been associated with the final candidate parent ion may be listed.
An alternative embodiment which involves a greater amount of data processing but yet which is intrinsically simpler is also contemplated. Once parent and daughter ions have been identified, then a parent ion mass chromatogram for each recognised parent ion is generated. The centre of each peak in the parent ion mass chromatogram and the corresponding parent ion elution time(s) are then determined. Similarly, a daughter ion mass chromatogram for each recognised daughter ion is generated, and the centre of each peak in the daughter ion mass chromatogram and the corresponding daughter ion elution time(s) are then determined. Rather than then identifying only a sub-set of the recognised parent ions, all (or nearly all) of the recognised parent ions are then identified. Daughter ions are assigned to parent ions according to the closeness of fit of their respective elution times and all daughter ions which have been associated with a parent ion may then be listed.
Although not essential to the present invention, ions generated by the ion source may be passed through a mass filter, preferably a quadrupole mass filter, prior to being passed to the fragmentation means. This presents an alternative or an additional method of recognising a daughter ion. A daughter ion may be recognised by recognising ions in a high fragmentation mass spectrum which have a mass to charge ratio which is not transmitted by the fragmentation means i.e. daughter ions are recognised by virtue of their having a mass to charge ratio falling outside of the transmission window of the mass filter. If the ions would not be transmitted by the mass filter then they must have been produced in the fragmentation means.
The ion source may be either an electrospray, atmospheric pressure chemical ionization or matrix assisted laser desorption ionization (xe2x80x9cMALDIxe2x80x9d) ion source. Such ion sources may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of liquid chromatography or capillary electrophoresis.
Alternatively, the ion source may be an electron impact, chemical ionization or field ionisation ion source. Such ion sources may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of gas chromatography.
A mass filter, preferably a quadrupole mass filter, may be provided upstream of the collision cell. However, a mass filter is not essential to the present invention. The mass filter may have a highpass filter characteristic and, for example, be arranged to transmit ions having a mass to charge ratio selected from the group comprising: (i) xe2x89xa6100; (ii) xe2x89xa6150; (iii) xe2x89xa6200; (iv) xe2x89xa6250; (v) xe2x89xa6300; (vi) xe2x89xa6350; (vii) xe2x89xa6400; (viii) xe2x89xa6450; and (ix) xe2x89xa6500. Alternatively, the mass filter may have a lowpass or bandpass filter characteristic.
Although not essential, an ion guide may be provided upstream of the collision cell. The ion guide may be either a hexapole, quadrupole or octapole.
Alternatively, the ion guide may comprise a plurality of ring electrodes having substantially constant internal diameters (xe2x80x9cion tunnelxe2x80x9d) or a plurality of ring electrodes having substantially tapering internal diameters (xe2x80x9cion funnelxe2x80x9d).
The mass analyser is preferably either a quadrupole mass filter, a time-of-flight mass analyser (preferably an orthogonal acceleration time-of-flight mass analyser), an ion trap, a magnetic sector analyser or a Fourier Transform Ion Cyclotron Resonance (xe2x80x9cFTICRxe2x80x9d) mass analyser.
The collision cell may be either a quadrupole rod set, a hexapole rod set or an octopole rod set wherein neighbouring rods are maintained at substantially the same DC voltage, and a RF voltage is applied to the rods. The collision cell preferably forms a substantially gas-tight enclosure apart from an ion entrance and ion exit aperture. A collision gas such as helium, argon, nitrogen, air or methane may be introduced into the collision cell.
In a first mode of operation (i.e. high fragmentation mode) a voltage may be supplied to the collision cell selected from the group comprising: (i) xe2x89xa715V; (ii) xe2x89xa720V; (iii) xe2x89xa725V; (iv) xe2x89xa730V; (v) xe2x89xa750V; (vi) xe2x89xa7100V; (vii) xe2x89xa7150V; and (viii) xe2x89xa7200V. In a second mode of operation (i.e. low fragmentation mode) a voltage may be supplied to the collision cell selected from the group comprising: (i) xe2x89xa65V; (ii) xe2x89xa64.5V; (iii) xe2x89xa64V; (iv) xe2x89xa63.5V; (v) xe2x89xa63V; (vi) xe2x89xa62.5V; (vii) xe2x89xa62V; (viii) xe2x89xa61.5V; (ix) xe2x89xa61V; (x) xe2x89xa60.5V; and (xi) substantially OV. However, according to less preferred embodiments, voltages below 15V may be supplied in the first mode and/or voltages above 5V may be supplied in the second mode. For example, in either the first or the second mode a voltage of around 10V may be supplied. Preferably, the voltage difference between the two modes is at least 5V, 10V, 15V, 20V, 25V, 30V, 35V, 40V, 50V or more than 50V.